Introduction to CyTOF

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Characterizing large quantities of single cells is important to address clinically and biologically relevant questions.

Traditionally, bulk measurements have been used to characterize the functional states of diseased vs. normal immune cells, livers, brains, and tissues or organs of interest to identify various interesting mechanistic differences. However, these bulk measurements average information across many cells, thereby masking potentially interesting and relevant heterogeneity. Ideally, measurements should be made with single cell resolution. Additionally, cells do not work alone but rather together, responding to and compensating for each other. Particularly in the context of disease vs. normal, often the proportion of various subpopulations relative to each other is more critical to the behavior of subpopulations in the diseased vs. normal tissue. Rare novel subpopulations may also be of interest. Therefore, measurements should be made on, not just single cells, but a large quantity of single cells.


Historically, fluorescence-based flow cytometry has historically been the premier tool for such high-throughput single cell analysis.

In fluorescence-based flow cytometry, single cells are stained with flourophore conjugated antibodies that bind to cell surface proteins. Cells are suspended in a stream of fluid and passed through a laser that excites the flourophores, resulting in emission of photons in a range of wavelengths (ie. color) that can then be detected and quantified. Therefore, by detecting the colors emitted by flourophore, we can effectively recover the antibody and subsequently protein of interest. However, we can only detect as many proteins or markers as we have discernible colors. As such, fluorescence-based flow cytometry is limited by fluorescence spectral overlap and only a handful of proteins or markers may be characterized per cell.

Another method for characterizing samples, inductively coupled plasma mass spectrometry (ICP-MS), resolves and quantifies elemental components.

In ICP-MS, a sample is atomized and ionized (ie. transformed into electrically charged particles) in hot plasma and shot through a magnetic field. The magnetic field exert forces on these ions, deflecting the ions onto a detector. The magnitude of the deflection depends on the ion’s mass-to-charge ratio. Lighter ions get deflected by the magnetic force more than heavier ions based on Newton’s second law of motion, F = ma. Each ion has a known associated mass and location on the detector. Therefore, by inspecting the peaks on the detector, we can identify and quantify the ions in our sample.


Single cell mass cytometry, CyTOF (Cytometry Time Of Flight), combines concepts from flourescence-based flow cytometry and ICP-MS.

Like in flourescence-based flow cytometry, rather than staining single cells with antibodies conjugated fluorophores, cells are stained with antibodies are conjugated to rare (not found in normal biological systems) transition element isotopes of unique mass. Like in ICP-MS, cells are sprayed into plasma and shot through a magnetic field and the resulting rare transition element isotope ions are detected and quantified. In this manner, by detecting the rare transition element isotope ion, we can effectively recover the antibody and subsequently protein of interest. Unlike flourophores, elemental masses do not produce overlapping spectrums and thus we are limited only by the number of rare stable isotopes available and compatible with the metal chelating polymer chemistry used to attach them to antibodies. Currently, approximately 40 such rare stable isotopes are available.


CyTOF is most appropriate for characterizing large number of single cells based on relatively few markers and preservation of cell is not needed.

Like fluorescence-based flow cytometry, CyTOF is capable of characterizing millions of single cells. Compared to fluorescence-based flow cytometry, CyTOF cannot detect cellular calcium and reactive oxygen species. Most notably, in CyTOF, cells are destroyed in plasma and cannot be recovered for further downstream analysis. Other single cell methods such as targeted single cell rt-qPCR methods can do 96 transcripts but is limited to 96 cells per plate. Similarly, single cell RNA-seq can characterize, in theory, the entire transcriptome, identify alternative splicing, exon-based mutations but is limited to 96 cells per plate. Thus, CyTOF is most appropriate for characterizing a large number of single cells based on relatively few markers and where preservation of cell is not needed.

The future and potential of CyTOF lies in combining it with other methods.

CyTOF is expensive in terms of the machine, the technician needed to operate the machine, the chemistry, the antibodies, the testing of antibodies, etc. CyTOF may most effectively be used to identify rare subpopulations by surface markers that can then be isolated using fluorescence-activated cell sorting (FACs) and analyzed further using growth assays, single cell DNA, RNA-seq, or ATAC-seq, or other methods.


So, what do you think ?